Immunocytochemical studies on the vimentin distribution and cell proliferation of fibroblasts in patients with Friedreich's ataxia.

Fibroblasts obtained from patients with Friedreich's ataxia and normal control subjects were studied by immunocytochemistry for intermediate filament vimentin and also for in vitro proliferation. Trypsinized cells were seeded on coverslips and incubated for 1.5 h and 24 h. The expression of vimentin in cells was investigated by immunofluorescence microscopy. Cell proliferation was studied with BrdU antibody technique. Cells from patients with Friedreich's ataxia showed a slower outgrowth of vimentin filaments in comparison to cells from normal controls. These cells also incorporated less 5-bromo-2'-deoxyuridine (BrdU) into their DNA. The observations may be relevant to the clinical manifestations of the disease which involves many organs in addition to brain and spinal cord.

[Investigation of a variant form of hypoxanthine-phosphoribosyl transferase in a family (author's transl)]. / Untersuchung einer varianten Form der Hypoxanthin-Phosphoribosyl-Transferase in einer Familie.

In a family study with two patients showing hyperuricaemia, discrete neurological symptoms, as well as gouty arthritis in the older proband, hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) were determined in haemolysates and fibroblast extracts. 6 normal subjects and 3 patients with the Lesch-Nyhan syndrome were examined as controls. Reduced HPRT activity by 18% and 12% in the proband and his nephew, respectively, together with an increase to values of 206% and 113% in APRT activity was observed in haemolysates. In fibroblasts the HPRT activity was reduced to 40--43%, but the APRT activity was within the normal range. The studies indicate the presence of a possible variant form of HPRT in these patients.

Expression, regulation and clinical relevance of the ATPase inhibitory factor 1 in human cancers.

Recent findings in colon cancer cells indicate that inhibition of the mitochondrial H(+)-adenosine triphosphate (ATP) synthase by the ATPase inhibitory factor 1 (IF1) promotes aerobic glycolysis and a reactive oxygen species (ROS)-mediated signal that enhances proliferation and cell survival. Herein, we have studied the expression, biological relevance, mechanism of regulation and potential clinical impact of IF1 in some prevalent human carcinomas. We show that IF1 is highly overexpressed in most (>90%) of the colon (n=64), lung (n=30), breast (n=129) and ovarian (n=10) carcinomas studied as assessed by different approaches in independent cohorts of cancer patients. The expression of IF1 in the corresponding normal tissues is negligible. By contrast, the endometrium, stomach and kidney show high expression of IF1 in the normal tissue revealing subtle differences by carcinogenesis. The overexpression of IF1 also promotes the activation of aerobic glycolysis and a concurrent ROS signal in mitochondria of the lung, breast and ovarian cancer cells mimicking the activity of oligomycin. IF1-mediated ROS signaling activates cell-type specific adaptive responses aimed at preventing death in these cell lines. Remarkably, regulation of IF1 expression in the colon, lung, breast and ovarian carcinomas is exerted at post-transcriptional levels. We demonstrate that IF1 is a short-lived protein (t1/2 ∼100 min) strongly implicating translation and/or protein stabilization as main drivers of metabolic reprogramming and cell survival in these human cancers. Analysis of tumor expression of IF1 in cohorts of breast and colon cancer patients revealed its relevance as a predictive marker for clinical outcome, emphasizing the high potential of IF1 as therapeutic target.

Characterization of long-term cell cultures of human chorion villi and fibroblasts using antibodies to cytoskeletal proteins.

In long-term cultures of chorion villi the expression of cytoskeletal proteins--vimentin, cytokeratin, actin, tropomyosin and vinculin--was studied using immunoreactivity to antibodies. Vimentin was detected in all cells of chorionic villi cultures despite their morphological appearance. In comparison to fibroblast cultures from human skin biopsies with bundle-like vimentin structures, chorionic villi cells showed fine ramified vimentin filaments. Some of the chorionic villi cells with typical fibroblastoid appearance also had cytokeratin filaments. No differences were observed in the distribution of vinculin. Tropomyosin structure and actin filaments could be detected in cultures of chorionic villi whereas fibroblasts showed only an intense diffuse staining.

Two-dimensional electrophoresis of soluble and structure-bound proteins from cultured human fibroblasts and hair root cells: qualitative and quantitative variation.

Proteins from cultured human fibroblasts and native human hair root cells were investigated using the two-dimensional electrophoresis (2DE) technique. Cell material from 35 different healthy persons was examined. Proteins of different sources were separated: total proteins of fibroblasts (12 cell lines), soluble proteins of fibroblasts (12 cell lines), structure-bound proteins of fibroblasts (eight cell lines) and soluble proteins of hair root cells (12 subjects). The protein samples of different individuals were run in pairs through the electrophoresis procedure and the two patterns of each pair were compared. All changes in the electrophoretic mobility of polypeptide spots (qualitative variants) and all clearly visible differences in the staining intensity of the spots (quantitative variants) were scored. Less than 1% of the qualitative variants per pattern was found in total cell proteins and this percentage was not increased in soluble proteins. No qualitative variation was detected in structure-bound proteins. Quantitative variation occurred to a considerably higher degree in the 2DE patterns than qualitative changes. The incidence of quantitative variants was about three times higher in soluble proteins (11%) than in structure-bound proteins (3.5%); in the total cell proteins it lay in between (7%). Cultured cells (fibroblasts) and native cells (hair root cells) showed a similar degree of variation. A comparison of the data shown here with data obtained by an investigation on inbred strains of the mouse suggest that the major part of the quantitative variants observed in the 2DE patterns of proteins were genetically determined. The results presented here and the mouse data mentioned above lead us to the conclusion that the genetic variability of proteins may be characterized by quantitative changes rather than by qualitative changes, and that the genetic variability occurs to quite different degrees in different classes of proteins: structure-bound proteins less than soluble non-enzymatic proteins less than enzymes (certain groups).

Studies in fibroblasts of patients with the Lesch-Nyhan syndrome and HPRT variants. Correlation of HPRT activity with hypoxanthine utilization and growth in selection media.

In the present study, hypoxanthine phosphoribosyltransferase (HPRT) has been investigated in fibroblasts of 19 patients from 16 different families with HPRT deficiency, concerning activity, incorporation of 14C-hypoxanthine, and growth in 8-azaguanine and HAT (hypoxanthine, azaserine, thymidine containing) selection media. According to these data we could classify the patients into 5 groups (patients with classical Lesch-Nyhan syndrome and patients with HPRT variants of types A, B, C, D). In 3 groups (patients with classical Lesch-Nyhan syndrome, HPRT variants C and D), a correlation of residual HPRT activity with the incorporation of 14C-hypoxanthine as well as growth in 8-azaguanine and HAT selection was observed. The variant A, from a patient with the classical Lesch-Nyhan syndrome, exhibited higher HPRT activity than that from all the other patients with the Lesch-Nyhan syndrome. However, the values of hypoxanthine incorporation and growth in selection media were as in the classical syndrome. The cells of variant B were resistant to azaguanine and grew in HAT selection media in the range of control cells, but had HPRT residual activities similar to those of variants A and C. For the characterization of the genetic heterogeneity of HPRT, it seems necessary to study the enzymatic properties in cell extracts as well as the purine uptake and proliferation of cells in different selection media.

A rapid micromethod for prenatal diagnosis of Lesch Nyhan syndrome.

A method is described which enables prenatal diagnosis of Lesch Nyhan Syndrome (HGPRT deficiency) to be made within 7-10 days. The procedure is based on the direct cultivation of amniotic cells in microtest II plates; the HGPRT reaction is performed in individual wells containing between 500 to 10,000 cells, and is followed by separation of the radioactive reaction products by means of microchromatography on 3 cm x 5 cm PEI plates. This method permits determination of the actual HGPRT enzyme activity of the cell lines.

A rapid micro culture method of chromosome preparations from fibroblasts and amniotic cells.

A micromethod enabling the making of chromosome preparations from fibroblasts and amniotic cells cultured on surface-treated Microtest II plates is presented. Preparations from each well contain sufficient metaphases to perform a reliable diagnosis. The potential for the use of this method in early prenatal diagnosis is discussed.

Growth studies on fibroblasts of patients with autosomal recessive Friedreich's ataxia.

Fibroblasts derived from patients with undisputed autosomal recessive Friedreich's ataxia were examined for their growth characteristics including plating efficiency, proliferation curves and cumulative number of population doublings. In comparison to cells of healthy controls, the cells from ataxia patients showed lower plating efficiency, growth rate and number of cumulative population doublings in these parameters. Cells of heterozygotes showed clonal growth and growth curves in the range of the healthy controls.

Heterogeneity in maple syrup urine disease: aspects of cofactor requirement and complementation in cultured fibroblasts.

Fibroblast strains derived from six patients with maple syrup urine disease have been investigated for their requirements of the cofactors NAD, CoASH, Mg++ and TPP in comparison with 10 normal control strains. The reconstitution of the decarboxylase function of branched chain alpha-keto acid (BCKA) dehydrogenase complex in lysed cells was studied with respect to the substrates alpha-keto-isocaproic acid, alpha-keto-isovaleric acid, and alpha-keto-beta-methylvaleric acid (KIC, KIVA, MEVA). The enzyme activity of all normal control strains for the substrates KIC and KIVA was not reconstituted by TPP + Mg++ alone, but CoASH + NAD could reconstitute the enzyme activity with KIC and KIVA in different degrees. Only two control strains were tested with MEVA as substrate, and these showed in contrast that TPP + Mg++ could partly reconstitute the enzyme activity. In contrast to the relative homogeneity in the reconstitution profiles of normal strains, the five classical and one intermittent MSUD strains showed heterogeneity in cofactor requirements. Complementation analysis using heterokaryons prepared from fibroblasts of four patients with classical MSUD and one patient with intermittent MSUD showed, in contrast to experiments with normal controls, a partial amelioration of the defect in two combinations; it is suggested that the defect in these strains is located at different functional subunits of the multienzyme complex.

High resolution protein mapping in fibroblast cell lines and hair roots from patients with genetic disease.

Protein patterns of cultured fibroblast and hair root lysates from healthy controls and patients with genetic diseases (Duchenne muscular dystrophy, Friedreich's ataxia, Marie's ataxia, Lesch-Nyhan syndrome, maple syrup urine disease, and trisomy 13, 18 and 21) were obtained with two-dimensional electrophoresis. The analysis of these patterns in 39 gels by visual comparison revealed differences in the presence and absence of 20 specific protein spots. However, this variability, which has been observed in healthy controls as well as in patients, could not provide a diagnosis for a specific genetic disease. Only in one case - trisomy 18 - was an additional spot observed, which was not present in any of the other gels.

Phenotypic interaction studies of HPRT mutant and normal human fibroblasts.

Hypoxanthine incorporation was studied in growing HPRT mutant cells by preincubating them with extracts from normal cells, HPRT mutant cells, and extracts of their lyophilized cell sediment. HPRT mutant cells showed no increase of hypoxanthine uptake after preincubation with extracts of mutant cells, whereas after preincubation with extracts from normal cells and lyophilized sediment of HPRT mutant cells the incorporation rate was increased. This effect could not be observed when normal cells were preincubated with extracts of lyophilized sediments of normal cell lines.

High-resolution protein mapping of human fibroblasts and hair root cells: a standardized reproducible procedure considering the effect of cell culture parameters.

The effect of different cell culture parameters on two-dimensional polypeptide maps of human fibroblasts is considered. Improved culture methods are introduced to get better resolution and reproducibility. Based on these investigations, highly standardized techniques for the protein mapping of this tissue and also of hair root cell lysates are presented. These methods seem to be quite suitable for analysis of cellular synthesized proteins and study of variability in normal subjects and in patients with genetic disorders.

Analysis of inborn errors of metabolism and other genetic defects in human fibroblasts using two-dimensional polypeptide mapping.

A standardized technique for the two-dimensional polypeptide mapping of cultured human fibroblasts has been used for the study of cellular protein variations in healthy controls, patients with inborn errors of metabolism and some other genetic defects. The analysis of about 50 gels has established that this method is very reproducible and enables the examination of about 600 polypeptides in a single gel. The inter-individual variation has been rather low (about 3%). In the gels from patients with genetic defects only very minor qualitative variations have been observed.

Characterisation of human tumour cell lines using antibodies to intermediate filaments.

The human hepatoma cell line G2 (Hep G2) has been compared to lung carcinoma, sarcoma and skin fibroblasts for the expression of intermediate filaments, i.e. vimentin and cytokeratin. The immunofluorescence study revealed that, in contradiction to Szecheng et al. (1987), cytokeratin and vimentin are absent in Hep G2. Human skin fibroblasts and sarcoma cells expressed vimentin as expected for their mesenchymal origin, but a positive reaction to vimentin could also be shown in lung carcinoma cells. However, the vimentin filament structure of both these tumour cell lines was different in comparison with skin fibroblasts. Therefore determining the exact tissue origin of tumour cell lines by means of intermediate filament characterization remains doubtful.